Thinlayerchromatography(TLC)
Biological Techniques

Introduction of TLC
 It is a technique to separate components of non-
volatile mixtures using a thin stationary phase on
some plates (glass)
 It may be used for analytical or preparatory scale
 It is performed on a sheet of glass, plastic or
aluminium foil which is coated with a thin layer
of adsorbent material like silica gel, aluminium
oxide (alumina) or cellulose
 This layer of adsorbent acts as the stationary phase

Introduction of TLC
 A mixture is separated on the basis of affinities for
the mobile and stationary phases
 A solvent (mobile phase) draws up the plate
via capillary action and separates components of a
mixture
 Different components rise TLC plate at different rates
which results in the separation
 Rate of upward movement of the sample on the plate
depends on how tightly the sample binds with the
stationary phase

Introduction of TLC
 Spots of the sample on the plate are visualized by
various methods
 Distance travelled by a component is measured as
compared with the distance covered by the solvent in
a given time
 This is named as retardation factor (Rf) which ranges
from zero to one
 This technique is being widely used due to its
simplicity, low cost, high sensitivity and fast speed

Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Principle

Principle of TLC
 Principle of TLC is similar to paper chromatography
 Different compounds in the sample travel at
different rates due to different affinities for the
stationary phase and the solvent
 Separation is based on the competition between the
solute and the mobile phase for binding places on
the stationary phase
 Rf values are used to identify the compounds
because of their uniqueness

Principle of TLC
 Rf is the distance travelled by
a component divided by the
distance by the solvent
 Rf values depend on polarity of
the components, stationary as
well as mobile phase
 More polar compound have
stronger interaction with the
silica (polar stationary phase)
as compared with the less
polar compounds
 As a consequence the less polar
compound moves higher up
the plate and have higher Rf
values

Principle of TLC
 For more polar mobile phase all compounds on TLC
plate move high up
 Generally a substance whose structure resembles to
that of the stationary phase will have low Rf value
 Rf values depend on layer thickness, moisture on the
TLC plate, temperature, nature of mobile phase,
nature of TLC plate, sample size, etc.
 Mobile phase must not be allowed to reach the end
of the stationary phase

Principle of TLC
 Rf values of the known compounds or standards are
calculated
 Unknown compounds are identified by comparing
with Rf values of the known ones
 Compounds which are more soluble in the mobile
phase (solvent) will travel high up on the plate
 Similarly compounds more similar to the stationary
phase will travel less on the plate than the different
ones during separation

Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Components

Components of TLC
Stationary phase
 TLC plates are prepared by mixing the adsorbent such
as silica or alumina with a small amount of inert
calcium sulfate and water
 This mixture is spread as a thick slurry on an
unreactive sheet
 A compound which fluoresces under UV may also be
used
 Plate is dried and activated by heating in an oven
 Choice of stationary phase depends on the sample

Mobile phase
 Selection of a good solvent system is very important
 TLC is started with a nonpolar solvent such as ligroin
followed by a polar solvent such as ether or ethyl
acetate
 Rf values should vary between zero to one
 Separation depends on solubility of the compound in
a solvent
 Acids, bases or polar compounds require special
solvent composition
Components of TLC

Spotting the plate
 Once the sample is prepared a spotting capillary is
used to add the sample to the plate
 Spotting capillaries must be extremely small
 simply touch the end of the capillary tube at the start
line of the plate
 Smearing, smudging and spots that overlap will result
in inefficient separation
 Solvent is allowed to evaporate
 Spots should be separate
Components of TLC

Rf values
 Rf value is retardation factor and it is the distance
traveled by the individual component as compared
with the solvent
 Components of a mixture may have different Rf
values
 Rf values of pure compounds is used as a reference
value
 Unknown compounds are identified by comparing
their Rf values
 Rf values depend on how tightly the sample binds to
the stationary phase
Components of TLC

Components of TLC

Spot visualization
 It is easy to visualize spots of colored compounds
 However, since most organic compounds are
colorless they are observed by UV light or by some
other methods
 TLC plates glow green under UV light due to presence
of fluorescent indicator
 Plates are also placed in jars containing iodine stain
which turns organic spots to brown
 Permanganate and other stains are also used
Components of TLC

Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Method

Sample application
 Different samples are applied in a row of spots at the
same distance from the bottom edge
 Each sample will move in its own lane from the starting
point
 Samples are applied just above the solvent layer so
that they are not dissolved
 A standard is also applied for the comparison
 A co-spot is also applied by applying the sample on the
standard
Method of TLC

Solvent and capillary action
 A small amount of solvent is poured in a container
 The container is closed with a cover glass to achieve
the equilibrium and to avoid the evaporation
 Solvent is used according to the sample being
analyzed
 Solvent is allowed to rise up the plate by capillary
action about one cm below the upper end of the
plate
 Solvent is never allowed to run the end of the plate
Method of TLC

Plate visualization
 TLC plates are pre-coated with a fluorescent material
such as zinc sulfide which allows some of the
compounds to be visualized when examined under UV
light
 Plates can also be visualized by applying other
chemicals after elution
 Various visualizing agents react with the spots to
produce different results
 Use of different agents depends on the types of
samples
Method of TLC

Thin layer
chromatography (TLC)-
Common problems
Advanced Analytical Techniques

Common problems in TLC
 Since length of the stationary phase is limited in TLC,
there may be limited separation as compared with
other methods
 It may not withstand high temperature required for
the separation
 Similarly strong acidic and oxidizing stains may also
damage the stationary phase
 There may be problems of contaminations during
processing and handling of the TLC plates
Stationary phase

Common problems in TLC
 Mobile phase may advance with uneven flow
 This may lead to inaccurate Rf values
 It may be avoided by placing TLC plates vertically
without any contact
 There should be enough solvent so that it can travel
upto most of length of plate
 TLC plate should be cut evenly for the separation
 Choice of a suitable solvent system is critical
Mobile phase

Common problems in TLC
 Concentrated samples may lead to streaks instead of
isolated spots
 This is due to limited separation by the mobile phase
 It may be avoided by diluting the sample for efficient
elution
 Samples should be applied above the solvent level
 Too big spots of samples on the TLC plate may also
lead to inadequate separation
 Samples should be spotted with care
Streaking

Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Applications

Measure of sample purity
 Purity of sample is analyzed by direct comparison of
the sample and the standard
 In case of impurities extra spots are detected on
visualization of the plates
Identification of compounds
 TLC can be used for purification, isolation and
identification of the natural products like alkaloids,
steroids, etc.
 Standards are used for the comparison
Applications of TLC

Analysis of reactions
 Reaction mixture is examined to assess whether
certain reaction is complete or not
 Formation of specific spots on the plates indicate
the presence of certain products
Biochemical analysis
 TLC is useful in separation of biochemical
metabolites from body fluids like blood plasma,
urine, etc.
 Other samples are also used to identify the
metabolites
Applications of TLC

Pharmaceutical industry
 TLC technique is used for detection of impurities in
the pharmaceutical products
 Various medicines like sedatives, antihistaminics,
analgesics, local anaesthetics, may be tested
qualitatively by TLC method
 It is also possible to analyze various intermediate
products formed in the development of
pharmaceutical products
 TLC can be applied in studying kinetics of the
products
Applications of TLC

Chemical analysis
 TLC is used for separation and identification of
various compounds closely related to each other
 It is also used in the identification of cations and
anions in inorganic chemistry
Food and cosmetics
 TLC method is used for separation and identification
of colors, preservatives, etc.
 It is also used in analysis of various cosmetic
products
Applications of TLC